The present invention relates generally to the examination of biological materials using optical spectroscopy and relates more particularly to a novel technique for examining biological materials using diffuse reflectance spectroscopy and the Kubelka-Munk function.
Optical spectroscopy has received increasing attention over the past several years as a tool for use in examining tissues and other biological materials. One such application of optical spectroscopy to the examination of tissues has been in the detection of cancer and precancerous states and has involved the use of native fluorescence spectroscopy. For example, in U.S. Pat. No. 4,930,516 inventors Alfano et al., which issued Jun. 5, 1990, and which is incorporated herein by reference, there is disclosed a method and apparatus for detecting the presence of cancerous tissue using visible luminescence. According to the aforementioned patent, the tissue to be examined is excited with a beam of monochromatic light that causes the tissue to fluoresce over a spectrum of wavelengths. The monochromatic light disclosed in the patent has a wavelength in the range of 350-500 nm. The intensity at which the excited tissue fluoresces is measured either over a spectrum or at a predetermined number of preselected wavelengths, such as at 531 nm, 522 nm and 633 nm. The patent further teaches that one can then determine the carcinomatoid status of the tissue in question by comparing the detected spectrum, one or more peak wavelengths of the detected spectrum, or a ratio or difference of particular wavelengths from the detected spectrum to standards obtained from known tissues.
Other patents and publications that relate to the use of native fluorescence in the detection of cancer and precancerous states include the following: U.S. Pat. No. 5,348,018, inventors Alfano et al., which issued Sep. 20, 1994; U.S. Pat. No. 5,467,767, inventors Alfano et al., which issued Nov. 21, 1995; U.S. Pat. No. 5,131,398, inventors Alfano et al., which issued Jul. 21, 1992; U.S. Pat. No. 5,042,494, inventor Alfano, issued Aug. 27, 1991; U.S. Pat. No. 5,413,108; inventor Alfano, issued May 9, 1995; U.S. Pat. No. 5,769,081, inventors Alfano et al., issued Jun. 23, 1998; U.S. Pat. No. 5,612,540, inventors Richards-Kortum et al., issued Mar. 18, 1997; U.S. Pat. No. 4,957,114, inventors Zeng et al., issued Sep. 18, 1990; Alfano et al., xe2x80x9cLight sheds light on cancer-distinguishing malignant tumor from benign tissue and tumors,xe2x80x9d The Bulletin of the New York Academy of Medicine, 67:143-50 (1991); Yang et al., xe2x80x9cOptical Spectroscopy of Benign and Malignant Breast Tissue,xe2x80x9d Lasers in the Life Sciences, 7(2):115-27 (1996); Yang et al., xe2x80x9cFundamental Differences of Excitation Spectrum between Malignant and Benign Breast Tissues,xe2x80x9d Photochemistry and Photobiology, 66(4):518-22 (1997); Yang et al., xe2x80x9cExcitation Spectrum of Malignant and Benign Breast Tissues: A Potential Optical Biopsy Approach,xe2x80x9d Lasers in the Life Sciences, 7(4):249-65 (1997); Galeotti et al., xe2x80x9cOn the Fluorescence of NAD(P)H in Whole-Cell Preparations of Tumours and Normal Tissues,xe2x80x9d Eur. J. Biochem., 17:485-96 (1970); and Japanese Patent Application No. Sho-57-795, published Jul. 15, 1983, all of which are incorporated herein by reference.
In addition, it should be noted that native fluorescence has also been used to detect a number of other abnormal or disease states unrelated to cancer, such as the detection of caries in teeth (U.S. Pat. No. 4,479,499, inventor Alfano, which issued Oct. 30, 1984, and which is incorporated herein by reference) and the detection of atherosclerotic plaque in arteries (U.S. Pat. No. 4,913,142, inventors Kittrell et al., issued Apr. 3, 1990, and which is incorporated herein by reference).
Still another type of spectroscopic technique that has been used to examine tissues has involved the use of Raman spectroscopy. One such application of Raman spectroscopy to the examination of tissues has been in the detection of cancer and is exemplified by U.S. Pat. No. 5,261,410, inventors Alfano et al., which issued Nov. 16, 1993, and which is incorporated herein by reference. In the aforementioned patent, there is disclosed a method for determining if a tissue is a malignant tumor tissue, a benign tumor tissue, or a normal or benign tissue. The method is based on the discovery that, when irradiated with a beam of infrared monochromatic light, malignant tumor tissue, benign tumor tissue, and normal or benign tissue produce distinguishable Raman spectra. For human breast tissue, some salient differences in the respective Raman spectra are the presence of four Raman bands at a Raman shift of about 1078, 1300, 1445 and 1651 cmxe2x88x921 for normal or benign tissue, the presence of three Raman bands at a Raman shift of about 1240, 1445 and 1659 cmxe2x88x921 for benign tumor tissue, and the presence of two Raman bands at a Raman shift of about 1445 and 1651 cmxe2x88x921 for malignant tumor tissue. In addition, it was discovered that for human breast tissue the ratio of intensities of the Raman bands at a Raman shift of about 1445 and 1659 cmxe2x88x921  is about 1.25 for normal or benign tissue, about 0.93 for benign tumor tissue, and about 0.87 for malignant tumor tissue.
In addition, as exemplified by U.S. Pat. No. 5,293,872, inventors Alfano et al., which issued Mar. 15, 1994, and which is incorporated herein by reference, Raman spectroscopy has also been used to distinguish between calcified atherosclerotic tissue and fibrous atherosclerotic tissue or normal cardiovascular tissue.
Still yet another type of spectroscopic technique that has been used to examine tissues has involved the use of diffuse reflectance spectroscopy. An example of the application of diffuse reflectance spectroscopy to the examination of tissues is disclosed in U.S. Pat. No. 4,423,736, inventors DeWitt et al., which issued Jan. 3, 1994, and which is incorporated herein by reference. In the aforementioned patent, there is disclosed a method for evaluating erythema (i.e., the reddening of the skin, for example, by sunburn) utilizing skin reflection measurements, said method involving the use of diffuse reflectance spectra within the range of 400 to 700 nm.
Other patents and publications of interest include U.S. Pat. No. 5,369,496, inventors Alfano et al., which issued Nov. 29, 1994; U.S. Pat. No. 5,303,026, inventors Strobl et al., which issued Apr. 12, 1994; Zhadin et al., xe2x80x9cEnhancement of the Fluorescence Cancer Diagnostic Method of Tissues Using Diffuse Reflectance and the Analysis of Oxygenation State,xe2x80x9d SPIE Proceeding, 2697:142-8 (1996); and Bigio et al., xe2x80x9cInvited: Elastic scattering spectroscopy for diagnosis of tissue pathologies,xe2x80x9d OSA Tops on Biomedical Optical Spectroscopy and Diagnostics 1996, Vol. 3, pp. 14-19, edited by Eva Sevick-Muraca and David Benaron, all of which are incorporated herein by reference.
It is an object of the present invention to provide a new technique for examining a biological material, such as a biological tissue or cell.
It is yet another object of the present invention to provide a technique as described above that can be used for either in vivo or in vitro examination of a biological material.
It is still yet another object of the present invention to provide a technique as described above that can be used to distinguish benign tumors, like fibroadenomas, and pre-cancer from malignant tumors.
It is still another object of the present invention to provide a technique as described above that can be used to distinguish invasive carcinoma or mixed invasive and in situ from malignant tumor.
In furtherance of the above objects, as well as other objects described below or apparent from the description below, there is provided herein a method for determining whether a tissue or cell sample is cancerous, precancerous or not, said method comprising the steps of: (a) measuring the diffuse reflectance from the tissue or cell sample at at least a first wavelength and a second wavelength, wherein said first wavelength is a wavelength selected from the group consisting of 255-265 nm and wherein said second wavelength is a wavelength selected from the group consisting of 275-285 nm; (b) using the Kubelka-Munk function to transform the diffuse reflectance measurement obtained at said first and second wavelengths; and (c) using said transformed Kubelka-Munk measurement at said first and second wavelengths to determine whether or not the tissue or cell sample is cancerous.
According to another aspect of the invention, there is provided herein a method for examining a tissue or cell sample, said method comprising the steps of (a) obtaining a diffuse reflectance spectrum for the tissue or cell sample over a spectral range of approximately 250 to 650 nm, preferably approximately: 250 to 300 nm; (b) transforming said diffuse reflectance spectrum using the Kubelka-Munk function; and (c) using the transformed Kubelka-Munk function from diffuse reflectance spectrum to determine the levels of constitutents in the tissue or cell sample for which the Kubelka-Munk function is indicative. For example, the transformed Kubelka-Munk function from diffuse reflectance spectrum at about 260 nm, at about 280 nm and at about 480 nm may be used to determine the levels of DNA, protein and fat, respectively, in the tissue or cell sample for use in separating cancer from benign, normal and fibroadenoma tissue. These tissues include GYN, breast, GI, colon and cervix.
It is even yet another object of the present invention to provide a new apparatus for examining a tissue or cell in accordance with the above method.
Additional objects, as well as features, advantages and aspects of the present invention, will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which is shown by way of illustration specific embodiments for practicing the invention. These embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.